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The endonuclease domain of the LINE-1 ORF2 protein can tolerate multiple mutations.

Kines KJ, Sokolowski M, deHaro DL, Christian CM, Baddoo M, Smither ME, Belancio VP - Mob DNA (2016)

Bottom Line: Some of these mutations were found in residues which were predicted to be phosphorylation sites for cellular kinases.We mutated several of these putative phosphorylation sites in the ORF2 endonuclease domain and investigated the effect of these mutations on the function of the full-length ORF2 protein and the endonuclease domain (ENp) alone.Most of the single and multiple point mutations that were tested did not significantly impact expression of the full-length ORF2p, or alter its ability to drive Alu retrotransposition.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural and Cellular Biology, Tulane School of Medicine, Tulane Cancer Center and Tulane Center for Aging, New Orleans, LA 70112 USA.

ABSTRACT

Background: Approximately 17 % of the human genome is comprised of the Long INterspersed Element-1 (LINE-1 or L1) retrotransposon, the only currently active autonomous family of retroelements. Though L1 elements have helped to shape mammalian genome evolution over millions of years, L1 activity can also be mutagenic and result in human disease. L1 expression has the potential to contribute to genomic instability via retrotransposition and DNA double-strand breaks (DSBs). Additionally, L1 is responsible for structural genomic variations induced by other transposable elements such as Alu and SVA, which rely on the L1 ORF2 protein for their propagation. Most of the genomic damage associated with L1 activity originates with the endonuclease domain of the ORF2 protein, which nicks the DNA in preparation for target-primed reverse transcription.

Results: Bioinformatic analysis of full-length L1 loci residing in the human genome identified numerous mutations in the amino acid sequence of the ORF2 endonuclease domain. Some of these mutations were found in residues which were predicted to be phosphorylation sites for cellular kinases. We mutated several of these putative phosphorylation sites in the ORF2 endonuclease domain and investigated the effect of these mutations on the function of the full-length ORF2 protein and the endonuclease domain (ENp) alone. Most of the single and multiple point mutations that were tested did not significantly impact expression of the full-length ORF2p, or alter its ability to drive Alu retrotransposition. Similarly, most of those same mutations did not significantly alter expression of ENp, or impair its ability to induce DNA damage and cause toxicity.

Conclusions: Overall, our data demonstrate that the full-length ORF2p or the ENp alone can tolerate several specific single and multiple point mutations in the endonuclease domain without significant impairment of their ability to support Alu mobilization or induce DNA damage, respectively.

No MeSH data available.


Related in: MedlinePlus

Mutations in the ORF2 endonuclease domain from full-length L1 loci in the human genome. Bioinformatic analysis using L1Base [36] revealed numerous mutations in the ORF2 endonuclease domains of 134 intact, full-length L1 loci. Positions of mutations relative to the sequence of the L1.3 ORF2 endonuclease domain are indicated by a blue square above the amino acid residue
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Fig1: Mutations in the ORF2 endonuclease domain from full-length L1 loci in the human genome. Bioinformatic analysis using L1Base [36] revealed numerous mutations in the ORF2 endonuclease domains of 134 intact, full-length L1 loci. Positions of mutations relative to the sequence of the L1.3 ORF2 endonuclease domain are indicated by a blue square above the amino acid residue

Mentions: Using L1Base [36], we analyzed the sequences of full-length L1 loci present in the human genome to identify naturally occurring mutations in the ORF2 endonuclease domain. Our search parameters were set to identify L1 loci that contain intact ORF1 and ORF2 sequences (no gaps, premature stops or frameshifts). We identified 134 L1 loci that satisfy these search criteria, the majority of which were L1Ta and L1PA2 families (Additional file 1: Table S1). None of the 134 full-length L1 loci fitting our search criteria had any mutations at amino acid H230 and only one locus contained a mutation at D205 (Fig. 1; Additional file 1: Table S1). These two residues are absolutely critical for ORF2 endonuclease function [8, 31]. The maximum number of mutations found in the endonuclease domain of any of the 134 full-length L1 loci was 11. Aligning the ORF2 protein sequences of the full-length L1 loci extracted from L1Base against the active human L1.3 element revealed that 118 of the 239 amino acids in the endonuclease domain were mutated at least once (Fig. 1; Additional file 2: Figure S1). However, three of these mutations may be specific to the L1PA2 sequence (I15V, A21P, V208L).Fig. 1


The endonuclease domain of the LINE-1 ORF2 protein can tolerate multiple mutations.

Kines KJ, Sokolowski M, deHaro DL, Christian CM, Baddoo M, Smither ME, Belancio VP - Mob DNA (2016)

Mutations in the ORF2 endonuclease domain from full-length L1 loci in the human genome. Bioinformatic analysis using L1Base [36] revealed numerous mutations in the ORF2 endonuclease domains of 134 intact, full-length L1 loci. Positions of mutations relative to the sequence of the L1.3 ORF2 endonuclease domain are indicated by a blue square above the amino acid residue
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4837594&req=5

Fig1: Mutations in the ORF2 endonuclease domain from full-length L1 loci in the human genome. Bioinformatic analysis using L1Base [36] revealed numerous mutations in the ORF2 endonuclease domains of 134 intact, full-length L1 loci. Positions of mutations relative to the sequence of the L1.3 ORF2 endonuclease domain are indicated by a blue square above the amino acid residue
Mentions: Using L1Base [36], we analyzed the sequences of full-length L1 loci present in the human genome to identify naturally occurring mutations in the ORF2 endonuclease domain. Our search parameters were set to identify L1 loci that contain intact ORF1 and ORF2 sequences (no gaps, premature stops or frameshifts). We identified 134 L1 loci that satisfy these search criteria, the majority of which were L1Ta and L1PA2 families (Additional file 1: Table S1). None of the 134 full-length L1 loci fitting our search criteria had any mutations at amino acid H230 and only one locus contained a mutation at D205 (Fig. 1; Additional file 1: Table S1). These two residues are absolutely critical for ORF2 endonuclease function [8, 31]. The maximum number of mutations found in the endonuclease domain of any of the 134 full-length L1 loci was 11. Aligning the ORF2 protein sequences of the full-length L1 loci extracted from L1Base against the active human L1.3 element revealed that 118 of the 239 amino acids in the endonuclease domain were mutated at least once (Fig. 1; Additional file 2: Figure S1). However, three of these mutations may be specific to the L1PA2 sequence (I15V, A21P, V208L).Fig. 1

Bottom Line: Some of these mutations were found in residues which were predicted to be phosphorylation sites for cellular kinases.We mutated several of these putative phosphorylation sites in the ORF2 endonuclease domain and investigated the effect of these mutations on the function of the full-length ORF2 protein and the endonuclease domain (ENp) alone.Most of the single and multiple point mutations that were tested did not significantly impact expression of the full-length ORF2p, or alter its ability to drive Alu retrotransposition.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural and Cellular Biology, Tulane School of Medicine, Tulane Cancer Center and Tulane Center for Aging, New Orleans, LA 70112 USA.

ABSTRACT

Background: Approximately 17 % of the human genome is comprised of the Long INterspersed Element-1 (LINE-1 or L1) retrotransposon, the only currently active autonomous family of retroelements. Though L1 elements have helped to shape mammalian genome evolution over millions of years, L1 activity can also be mutagenic and result in human disease. L1 expression has the potential to contribute to genomic instability via retrotransposition and DNA double-strand breaks (DSBs). Additionally, L1 is responsible for structural genomic variations induced by other transposable elements such as Alu and SVA, which rely on the L1 ORF2 protein for their propagation. Most of the genomic damage associated with L1 activity originates with the endonuclease domain of the ORF2 protein, which nicks the DNA in preparation for target-primed reverse transcription.

Results: Bioinformatic analysis of full-length L1 loci residing in the human genome identified numerous mutations in the amino acid sequence of the ORF2 endonuclease domain. Some of these mutations were found in residues which were predicted to be phosphorylation sites for cellular kinases. We mutated several of these putative phosphorylation sites in the ORF2 endonuclease domain and investigated the effect of these mutations on the function of the full-length ORF2 protein and the endonuclease domain (ENp) alone. Most of the single and multiple point mutations that were tested did not significantly impact expression of the full-length ORF2p, or alter its ability to drive Alu retrotransposition. Similarly, most of those same mutations did not significantly alter expression of ENp, or impair its ability to induce DNA damage and cause toxicity.

Conclusions: Overall, our data demonstrate that the full-length ORF2p or the ENp alone can tolerate several specific single and multiple point mutations in the endonuclease domain without significant impairment of their ability to support Alu mobilization or induce DNA damage, respectively.

No MeSH data available.


Related in: MedlinePlus